Phytotaxa 494 (1): 042–058 ISSN 1179-3155 (print edition) https://www.mapress.com/j/pt/ PHYTOTAXA Copyright © 2021 Magnolia Press Article ISSN 1179-3163 (online edition) https://doi.org/10.11646/phytotaxa.494.1.2 Taxonomic and nomenclatural novelties in Leucoagaricus (Agaricaceae) from Brazil CELESTE HEISECKE1,2,4*, JAIME ANDRÉS DUQUE BARBOSA1,2,5, MARIA ALICE NEVES3,6 & ANIBAL ALVES DE CARVALHO JR1,2,7 1 Programa de pós-graduação em Botânica, Escola Nacional de Botânica Tropical, JBRJ, Rua Pacheco Leão 2040, Solar da Imperatriz, 22460-036, Rio de Janeiro, RJ, Brazil. 2 Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, Rua Pacheco Leão 915, 22460-030, Rio de Janeiro, RJ, Brazil. 3 Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Departamento de Botânica, Cidade Universitária, Trindade, 88040-900, Florianópolis, SC, Brazil. 4 �[email protected]; https://orcid.org/0000-0003-1316-2212 5 �[email protected]; https://orcid.org/0000-0001-9892-9245 6 �[email protected]; https://orcid.org/0000-0002-1810-4890 7 �[email protected]; https://orcid.org/0000-0002-6176-2039 *Corresponding author Abstract Leucoagaricus nzumbae sp. nov. (Agaricaceae) is described based on morphological and molecular data from Teresópolis, Rio de Janeiro, Brazil. This species is characterized by the delicate small basidiomes that turn completely lilac when dried, brownish color of the pileus surface, and trichodermal elements with intracellular and parietal brown pigments of the pileus covering. A lectotype and an epitype are designated for Leucoagaricus gongylophorus, a symbiotic fungus of leaf- cutting ants. Full description, nomenclatural notes, and illustrations of fresh basidiomes in situ and of the main macro-and micromorphological features of both species are provided. Keywords: Agaricales, Lepiota s.l., Tropics Introduction Leucoagaricus Locq. ex Singer (1948) includes widely distributed saprotrophic mushrooms that colonize soil, with greater number of species in the tropics than in the temperate zone (Vellinga 2004a). Around 135 species are known (He et al. 2019), which are characterized by the white to cream spore print, entire or slightly striate but not plicate pileus margin, free lamellae, metachromatic basidiospores in Cresyl blue, and absence of clamp connections and pseudoparaphyses (Singer 1948, 1986). Leucoagaricus is placed in Agaricaceae based on its agaricoid habit and basidiospore morphology (Singer 1948, 1986). Molecular studies show that the genus may not be monophyletic and is within Leucocoprinus Patouillard (1888) (Johnson & Vilgalys 1998, Johnson 1999, Vellinga 2004b, Vellinga et al. 2011). However, taxonomic, and phylogenetic relationships among some species of Leucoagaricus and Leucocoprinus remain unclear due to the large number of species in the clade and low representation in molecular studies (Vellinga 2004b, Vellinga et al. 2011). During fieldwork in the evergreen Atlantic Forest in Teresópolis, Rio de Janeiro, Brazil, two remarkable species of Leucoagaricus were found. Leucoagaricus nzumbae is proposed as a new species based on morphological and molecular data. Leucoagaricus gongylophorus (Möller 1893: 70) Singer (1986: 477) is a rarely fruiting and noteworthy species that is cultivated by attine ants (Attini, Formicidae, Hymenoptera) and is recorded for the first time for Rio de Janeiro State. In this paper, we provide taxonomic comments, georeferenced data for the collection locations and illustrations of both species. The typification of Leucoagaricus gongylophorus is discussed and proposed, and a sequence from a basidiome is provided for the first time for this species. 42 Accepted by Else Vellinga: 27 Jan. 2021; published: 31 Mar. 2021 Material and methods Sampling and morphological studies The collections included in this work were made in the Brazilian Atlantic Forest according to the methods laid out by Lodge et al. (2004). Macroscopic characters noted from fresh specimens follow Lodge et al. (2004) and the color annotations follow Kornerup & Wanscher (1978; e.g., 6C4). The specimens were dried using an electric dryer at 40°C for approximately 8h (Lodge et al. 2004). Microscopic studies were based on freehand sections of dry material, mounted on slides with water, 3% KOH, or 3% KOH plus Congo red, Cresyl blue or Melzer’s reagent (Largent 1977, Singer 1986). Photomicrographs were obteined using a Zeiss Axioskope equipped with AxioCam MRm digital camera and analyzed with the AxioVision Rel.4.6 software. At least 20 measurements of each microstructure were made for each specimen. Terminology follows Vellinga (1988) and Vellinga & Noordeloos (2001). The abbreviation L. is used for Lepiota (Pers.: Fr.) Gray (1821) and La. for Leucoagaricus. Q indicates the range of the quotient of length and width of the basidiospores (Largent et al. 1977, Vellinga 1988). The collections were deposited in the RB herbarium at the Jardim Botânico do Rio de Janeiro, Rio de Janeiro, Brazil, or otherwise noted. Herbarium abbreviations follow Thiers (continuously updated). DNA extraction, primers, PCR and sequencing A small piece of the fresh basidiomes was preserved in silica gel for DNA extraction, which was done using the CTAB method (Dentinger et al. 2010). Optimal dilutions of the DNA were used to amplify the internal transcribed spacer (ITS). PCR amplifications were made with the previously described primers ITS1F and ITS4 (White et al. 1990, Gardes & Bruns 1993) in an Eppendorf MasterCycler thermal cycler. The PCR products were cleaned and sequenced by Macrogen Inc. Sequence chromatograms were corrected using the software Geneious v.6.1.8 (http://www.geneious.com, Kearse et al. 2012) and the sequences were deposited in GenBank (accession numbers MT605380–MT605383). Phylogenetic analyses Our analysis included four newly generated ITS sequences and 77 were retrieved from GenBank (Table 1). The ingroup includes representatives of the Leucoagaricus/Leucocoprinus clade. The sequences chosen for comparison were based on results of BLAST search for the newly generated ITS sequences with zero e-value. In addition, were included sequences of Leucoagaricus species which exhibit a color change when damaged and dried (Liang et al. 2010; Vellinga 2010, Vellinga et al. 2010, Vizzini et al. 2017, Sysouphanthong et al. 2018, Latha et al. 2020), those related to La. gongylophorus and a number of attine ant cultivars from previous phylogenetic studies (Mueller et al. 1998, Vellinga 2004, Vellinga & Davis 2006, Ortiz et al. 2008, Vo et al. 2009, Muñoz et al. 2015), and some representative species of Leucoagaricus sect. Rubrotincti Singer (Ge et al. 2015, Hussain et al. 2018, Ullah et al. 2020). Also four ITS sequences from fungus ants’ gardens of Acromyrmex coronatus (Genbank accession number MN473881), Acromyrmex heyeri Forel (Genbank accession number KJ531208), Acromyrmex laticeps Emery (Genbank accession number AY642807), and Acromyrmex striatus Roger (Genbank accession number MK685751), each from a different Brazilian biome (Cerrado, Pampa, Amazonian, and Atlantic Forest, respectively) were incorporated. Four ITS sequences of Lepiota species were included in the ingroup because they are in the Leucoagaricus/Leucocoprinus clade even though the new combinations into Leucoagaricus have not yet been made (see Table I). Three sequences of Agaricus Linnaeus (1753) were selected as the outgroup. The alignments were made using MAFFT v. 7, with the E-INS-i strategy (Katoh & Standley 2013), and then manually adjusted with AliView (Larsson 2014). The dataset was divided into three data partitions: ITS1, 5.8S, ITS2. Maximum likelihood (ML) analyses were conducted using Standard RAxML version 8.2.11 (Stamatakis 2014). The ML analyses involved 100 ML searches, each one starting from one randomized step-wise addition parsimony tree, under a GTRGAMMA model, with all other parameters estimated by the software. Individual alpha-shape parameters, GTR rates, and empirical base frequencies will be estimated and optimized for each partition through the-q option. To verify the reliability of the nodes, multi-parametric bootstrapping replicates under the same model were computed, allowing the program to halt bootstrapping automatically using the autoMRE option (-f a-m GTRGAMMA-p-x-q- #autoMRE). A branch was considered strongly supported if it had bootstrap support (BS) ≥ 90% and moderately supported if it had BS ≥ 70%. TAXonomic in LeucOAGaricus (AGaricaceae) Phytotaxa 494 (1) © 2021 Magnolia Press • 43 TABLE 1. Specimens and sequences used for the molecular analyses. The sequences generated in this work are in bold. Taxon Voucher Country Genebank accession number Agaricus rufoaurantiacus JBSD126476 Dominican Republic MF511127 Agaricus rufoaurantiacus JBSD126479 Dominican Republic MF511135 Agaricus sp. CHC281 Brazil MT605383 Lepiota cf. abruptibulba PA156 Panama AF079732 Lepiota decorata L.L. Norvell 2051224 U.S.A. GU136197 Lepiota flammeotincta ecv3315 U.S.A. GU136165 Lepiota flammeotincta DUKE-JJ97 Costa Rica U85331 Lepiota fuliginescens ecv3219 U.S.A. GU136186 Lepiota oculata ecv3100 U.S.A. EU141947 Lepiota roseolivida ecv2990 U.S.A. EF121816 Lepiotaceae sp. BR006 Brazil EF527287 Lepiotaceae sp. PA341 Panama AF079747 Lepiotaceae sp. BR032 Brazil EF527311 Leucoagaricus adelphicus ecv2584 Holotype U.S.A. AY243623 Leucoagaricus adelphicus ecv2772 Paratype U.S.A. AY243624 Leucoagaricus amanitoides R.M. Davis Paratype U.S.A. EF080871 Leucoagaricus amazonicus A. Ortiz 162841 Holotype Colombia EU940371 Leucoagaricus asiaticus